The invention relates generally to the field of formulation of therapeutic proteins, and particularly to formulations for complexes of insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3).
Growth factors are polypeptides that stimulate a wide variety of biological responses (e.g. DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. A number of different growth factor families have been identified, including the transforming growth factor beta family (TGF-xcex2s), epidermal growth factor and transforming growth factor alpha (the TGF-xcex1s), the platelet-derived growth factors (PDGFs), the fibroblast growth factor family (FGFs) and the insulin-like growth factor family (IGFs), which includes IGF-I and IGF-II.
IGF-I and IGF-II (the xe2x80x9cIGFsxe2x80x9d) are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (kDa). IGF-I mediates the major effects of growth hormone, and is thus the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since the treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division) for cells in neural tissue.
Almost all IGF circulates in a non-covalently associated complex of IGF-I, insulin-like growth factor binding protein 3 (IGFBP-3) and a larger protein subunit termed the acid labile subunit (ALS), such that very little free IGF-I is detectable. The ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF-binding activity and appears to bind only to the IGF/IGFBP-3 complex (Baxter et al., J. Biol. Chem. 264(20):11843-11848, 1989), although some reports suggest that IGFBP-3 can bind to rat ALS in the absence of IGF (Lee et al., Endocrinology 136:4982-4989, 1995). The ternary complex of IGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa. This ternary complex is thought to act xe2x80x9cas a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGFxe2x80x9d (Blum et al. (1991), xe2x80x9cPlasma IGFBP-3 Levels as Clinical Indicatorsxe2x80x9d in MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS, pp. 381-393, E. M. Spencer, ed., Elsevier, N.Y.). While there is essentially no excess (unbound) IGFBP-3 in circulation, a substantial excess of free ALS does exist (Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).
The complex of IGF-I and IGFBP-3 (xe2x80x9cbinary complexxe2x80x9d or xe2x80x9cIGF-I/IGFBP-3xe2x80x9d) is considerably different from uncomplexed IGF-I, both physically and chemically. The binary complex is approximately 5 times larger than uncomplexed IGF-I, has a different overall pI, and has a different overall hydrophobicity. These differences cause the binary complex to behave quite differently than IGF-I.
Due to its wide range of activities, IGF-I has been developed as a treatment for a variety of conditions, including amyotrophic lateral sclerosis (commonly known as Lou Gehrig""s disease) and diabetes. Unfortunately, the administration of IGF-I is accompanied by a variety of undesirable side effects, including hypoglycemia, edema (which can cause Bell""s palsy, carpal tunnel syndrome, and a variety of other deleterious conditions), hypophosphatemia (low serum phosphorus), and hypernatermia (excessive serum sodium). Administration of IGF-I as a complex of IGF-I and IGFBP-3 can reduce or eliminate these undesirable side effects (Adams et al., 1996, Prog. Growth Factor Res. 6:2-4)
While administration of IGF-I/IGFBP-3 complex may be desirable, the complex, like many proteins, has very limited stability (shelf life) in most formulations. A variety of purportedly stable formulations have been disclosed for IGF-I, either alone or in combination with another proteins (e.g, growth hormone), but the formulations thus disclosed for IGF-I/IGFBP-3 have been unsatisfactory due to poor stability of the proteins. These formulations for binary complex require that the protein be frozen, frequently at very low temperatures (e.g., xe2x88x9270xc2x0 C.). Freezers, particularly the ultra-low temperature freezers required to maintain xe2x88x9270xc2x0 C., are uncommon outside of research facilities and are also very expensive. Accordingly, formulations which can be stored at normal refrigerator temperatures or higher while still providing a long shelf life are critical to the commercial development of IGF-I/IGFBP for use as a therapeutic.
A variety of formulations have been disclosed for IGF, particularly IGF-I. For example, U.S. Pat. No. 5,681,814 discloses an IGF-I formulation for use in subcutaneous administration which comprises IGF-I, 2-50 mg/ml of an osmolyte (e.g., sodium chloride), 1-15 mg/ml of a preservative (e.g. benzyl alcohol or phenol) in a buffered in solution at pH 5-5.5. International Patent Application No. WO 97/07816 discloses a liquid IGF-I formulation which comprises IGF-I and mannitol in a buffered solution. However, due to the substantial physico/chemical differences between IGF-I and IGF/IGFBP-3, there is no reasonable expectation that IGF-I formulations will be suitable for IGFBP-3.
It should be noted that, while IGFBP-3 is the most abundant of the IGF binding proteins (xe2x80x9cIGFBPsxe2x80x9d), at least five other distinct IGFBPs have been identified in various tissues and body fluids. Although these proteins bind IGFs, they originate from separate genes and have distinct amino acid sequences. Unlike IGFBP-3, other circulating IGFBPs are not saturated with IGFs. IGFBP-3 is the only IGFBP which can form the 150 kDa ternary complex with IGF and ALS. However, some of the other IGFBPs have also been suggested for use in combination with IGF-I as therapeutics.
However, despite the advantages of administering IGF-I as a complex with IGFBP-3, little has been disclosed regarding formulations useful for pharmaceutical applications. Bagi et al. (J. Bone Mineral Res. 9(8):1301-11311, 1994) disclose the administration of IGF-I/IGFBP-3 to ovariectomized rats. The IGF-I/IGFBP-3 complex was formulated in simple phosphate buffered saline (PBS). Celtrix Pharmaceuticals, Inc. has disclosed the use of IGF-I/IGFBP-3 formulated in acetate buffer (pH 5.5) with 105 mM sodium chloride (NaCl) as the osmolyte. However, this formulation is not ideal for a commercial pharmaceutical formulation, as it does not permit lyophilization of the product.
Lyophilization (freeze drying under controlled conditions) is commonly used for long term storage of proteins. The lyophilized protein is substantially resistant to degradation, aggregation, oxidation, and other degenerative processes while in the freeze dried state. The lyophilized protein is normally reconstituted with water optionally containing a bacteriostatic preservative (e.g., benzyl alcohol). Unfortunately, many preservatives (e.g., benzyl alcohol) are not compatible with proteins, or at least reduce stability. However, the addition of a preservative is currently recommended for drugs which will be administered for periods of 24 hours or more, and this recommendation may become a requirement for drugs sold in the U.S.
Acceptable commercial lyophilized pharmaceutical products must form an acceptable xe2x80x9clyo cakexe2x80x9d (mass of lyophilized product). Preferably the lyo cake has a smooth surface and uniform appearance. Lyophilized protein alone rarely makes an acceptable lyo cake, so suitable bulking agents must be added. Generally, carbohydrates such as mannitol, sorbitol, and sucrose are used as bulking agents in lyophilized pharmaceutical products. Additionally, a buffering agent is normally added, particularly in pharmaceutical formulations for proteins such as growth factors and cytokines. The buffering agent is used to control the pH of the formulation when it is in a liquid state (i.e., before lyophilization and after reconstitution) because proteins are normally particularly sensitive to pH fluctuations or extremes.
Accordingly, there is a need in the art for pharmaceutically acceptable formulations that provide high stability for IGF-I/IGFBP-3 drug products.
The inventors have created novel formulations for IGF-I/IGFBP-3 which provide long term stability for IGF-I/IGFBP-3 complex. The formulations of the instant invention are pharmaceutically acceptable.
The inventors have surprisingly found that pharmaceutical formulations of IGF-I/IGFBP-3 complex with very low levels of osmolyte salts are more stable than formulations with high levels of added salts. Additionally, the inventors have found the surprising and unexpected result that omission of pH buffer salts further increases the stability of IGF-I/IGFBP-3 formulations.
In a further surprising and unexpected discovery, the inventors have found that IGF-I/IGFBP-3 formulations with high protein concentrations and having low osmolyte salts and no added pH buffer salts have high stability.
In one embodiment, the formulations of the invention comprise IGF-I/IGFBP-3 complex, a bulking agent, and pH buffer salt. No added osmolyte salt is present in the formulations of this embodiment.
In a further embodiment, the formulations of the instant invention comprise IGF-I/IGFBP-3 complex and a bulking agent. No added osmolyte salts or pH buffer salts are present in the formulations of this embodiment. The formulations of this embodiment are particularly advantageous because they allow the preparation of pharmaceutical formulations which contain very high protein concentrations.
The formulations of the instant invention may be liquid formulations or lyophilized formulations. Optionally, they may also contain a non-ionic surfactant. Liquid formulations may optionally contain a preservative for reducing or eliminating bacterial growth.